One of the important strategies in securing our country's energy future lies in producing liquid transportation fuel from renewable cell-wall polymer biomass, Lignin is the second most abundant cell-wall biopolymer. While it is important for plant viability it hinders the degradation of the polysaccharide in the cell wall to simple fermentable sugars for ethanol production. Increased understanding of lignin biosynthesis and efficiently managing plant lignification will greatly facilitate the improvement of feedstock for efficient bioenergy/chemical production.
By comparative structure-function analysis, our studies have gained a detailed understanding of the basis for the regioselective O-methylation of lignin monomeric precursors and for other phenylpropanoids. With this information, we have generated a set of novel enzyme catalysts, namely, monolignol 4-O-methyltransferases (MOMTs). Expressing these novel catalysts in plants introduces non-natural lignin monomers that diminish lignin cross-linking and polymerization, and ultimately reduce lignin content in the cell wall. Particularly, we can create a 4-O-methyltransferase to preferentially methylate the G-lignin precursor, p-coniferyl alcohol, expression of which decreases the incorporation of G-lignin unit and thus alters the S/G ratio.
Lignin, the most abundant terrestrial biopolymer after cellulose, imparts structural integrity to the plant cell wall. However, its presence hinders the degradability of feedstock in biofuel production, thus lowering the biomass conversion efficiency. Despite significant progress in genetic and biochemical studies of the biosynthesis of monolignols, the source materials for lignin and lignans, the mechanism of lignification remains controversial. New methodologies or techniques to manipulate the structure of lignin would be useful for improving biofuel production. During industrial processing, lignin must be degraded to extract cellulose fibers to efficiently convert carbohydrates to liquid biofuel (Chapple, C., et al. (2007) Nat. Biotechnol. 25, 746-748; Somerville, C. (2006) Biofuel. Curr. Biol. 17, R115-R119).
Lignin precursors are exclusively O-methylated at their meta-positions (i.e., 3/5-OH) of the phenyl rings, and are precluded from the substitution at the para-hydroxyl position. In fact, the para-hydroxyls of monolignols are proposed to be important for generating oxidative radicals, cross-linking lignin units, and for storage of lignin precursors (through 4-O-glucosylation). Therefore, chemical modification, for instance methylation, of the para-hydroxyl (i.e., 4-OH) of monolignol is expected to interfere with the synthesis of the lignin polymer. To test this hypothesis, we employed a structure-based protein engineering approach, to investigate the molecular mechanisms of regiospecific O-methylation of lignin precursors and natural phenylpropenes, thereby, creating a set of novel monolignol 4-O-methyltransferases that will produce the non-natural para-methylated monolignols in plants. By expressing these engineered enzymes, we demonstrate the consequences of perturbing the natural lignin precursor pool, particularly in reducing the cross-linking and polymerization of lignin, thus lowering lignin content; meanwhile redirecting metabolic flux into the novel soluble- and the “wall-bound”-phenolic esters that are beneficial to plant health and the cell wall digestibility.
Specifically, we explored the structure-function relationships of two types of functionally distinct but structurally related enzymes, i.e., phenylpropene 4-O-methyltransferase and lignin 3/5-O-methyltransferase, to understand their distinctive regiospecific methylation and substrate discrimination. The resulting information was used to create comprehensive libraries of the variants of lignin 3/5-O-methyltransferase and phenylpropene 4-O-methyltransferase, employing both the approaches of structure-based rational design and the iterative site-directed saturation mutagenesis. With high-throughput colorimetric and/or isotopic functional screening, we selected a range of novel variants able to efficiently methylate the para-hydroxyl of monolignols. The best performing novel engineered monolignol 4-O-methyltransferases were expressed in plants to evaluate their effects on lignin content and composition.
The cell wall of plants represents the most abundant biomass on earth, and is the most promising source of renewable energy. After cellulose, lignin is the second major cell-wall biopolymer of vascular plants; it imparts mechanical strength to the stem and protects the cellulose fiber from chemical- and biological-degradation. Although lignin confers integrity and resistance to the cell wall, its presence there lowers the efficiency of using the cell wall's cellulosic biomass for energy production.
Lignin, a complex biopolymer of hydroxylated and methylated phenylpropane units, is mainly derived from the oxidative coupling of three different hydroxycinnamyl alcohols (or monolignols), i.e., p-coumaryl, coniferyl, and sinapyl alcohols, which differ from each other only by their degree of methoxylation (FIG. 1A). These three monolignols, incorporated into the lignin polymer, produce, respectively, p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) phenylpropanoid units. The G unit is singly methylated on the 3-hydroxyl group, whereas the S unit is methylated on both the 3- and 5-hydroxyl moieties. The ratio of S-to-G subunits dictates the degree of lignin condensation by allowing different types of polymeric linkages. Increased G content leads to highly condensed lignin composed of a greater portion of biphenyl and other carbon-carbon linkages, whereas S subunits are commonly linked through more labile ether bonds at the 4-hydroxyl position. In angiosperms, lignin is composed of guaiacyl and syringyl monomers, whereas gymnosperm lignin consists almost entirely of guaiacyl moieties. Either or both the reduction of lignin content and increasing the proportion of the more chemically labile S lignin are desirable as such changes would facilitate the degradation of the cell wall.
The biosynthesis of the lignin precursors proceeds through the common phenylpropanoid pathway, starting with the deamination of phenylalanine to cinnamic acid. Subsequent enzymatic reactions include the hydroxylation of the aromatic ring, the activation of cinnamic acids to cinnamoyl-CoA esters, the methylation of certain phenolic hydroxyl groups, and the reduction of the CoA esters to cinnamaldehydes and further to cinnamyl alcohol. The characterization of p-hydroxycinnamoyltransferase and p-coumarate 3-hydroxylase and the demonstration of hydroxylation and methylation reactions occurring preferentially at the cinnamaldehyde and cinnamyl alcohol level led to the significant revision and simplification of the proposed monolignol biosynthetic pathway.
Despite increasing knowledge of the enzymology of monolignol biosynthesis, the processes of plant lignification still are unclear, and the molecular mechanism of lignin polymerization remains controversial.
Monolignol 4-O-Glucosylation and Transport.
Monolignols are formed in the cytosol, after which they are sequestered into cell wall where subsequently they are polymerized to afford a wall-reinforcing biopolymer. The monolignol 4-O-β-D-glucopyranosides, i.e., E-coniferin and E-syringin, frequently accumulate in the cambial region of gymnosperms and some angiosperms. Long-standing hypothesis suggest that these monolignol glucosides may be storage reserves or transport forms of the monolignols, and that the uridine diphosphate glucose (UDPG)-coniferyl/sinapyl alcohol glucosyltransferases, together with coniferin-β-glucosidase may regulate the storage and mobilization of monolignols for lignan/lignin biosynthesis. A few UDPG-glycosyltransferases (UGTs) that 4-O-glucosylate coniferyl alcohol or sinapyl alcohol have been identified in Arabidopsis. Reverse genetic studies demonstrated the effects of disturbances in the formation of glycosidic monolignols. However, because UGTs comprise a large superfamily of enzymes that exhibit broad substrate specificity in vitro, it is difficult to precisely demonstrate their specific roles in monolignol 4-O-glycosylation and the roles of such glycosylation in the proposed lignin precursor transport, particularly when this assessment relies only on the reverse genetic approach.
Dehydrogenation of Monolignols and Phenoxy Radical-Radical Coupling.
After monolignols are transported to the cell wall, lignin is formed through oxidative dehydrogenation and subsequent coupling of the resulting phenoxy radicals. The dehydrogenation (single-electron oxidation) of monolignols is believed to be initiated at the para-hydroxyl (4-OH) site of the aromatic ring of the monolignol by oxidative enzymes (peroxidases/laccases), followed by electron delocalization (radical transfer), generating p-quinoid species, i.e., the free radical intermediates (as depicted with p-coniferyl alcohol in FIG. 1B).
The evidence that supports the involvement of the oxidases (peroxidases/laccases) in monolignol dehydrogenation is extensive but circumstantial. Genes for the peroxidases and laccases were cloned from various plant species and their expression and lignification response was examined. A few studies reported reduced lignin content as consequence of reduction in specific peroxidase and laccase expression.
The monolignol radicals generated by dehydrogenation are relatively stable owing to electron resonance, and are subsequently coupled to each other or to the growing polymers during lignification to form the lignin macromolecule. Much controversy has centered on the question of whether phenoxy radical cross-coupling is tightly controlled under protein/template guidance, or is a random chemical process. The cross-coupling of monolignols in lignin polymerization generates different inter-unit bonding, including the most frequent β-O-4 (β-aryl ether) linkage, and less frequent 5-O-4 ether linkage (FIG. 1B); formation of these ether linkages between phenylpropane units directly involves the un-substituted 4-hydroxyl positions.
Reverse Genetic Studies on Lignin Biosynthesis.
Transgenic approaches have been intensively employed to explore the in vivo functions of lignin biosynthetic genes and enzymes. They have produced a growing knowledge of monolignol biosynthesis and also appear to have great biotechnological potential for manipulating plant lignification. Almost all genes encoding the enzymes in monolignol biosynthetic pathways have been down-regulated in different plant species. In many cases, the gene down-regulation reduced lignin content or changed its composition. Interestingly, in a few cases, repressing lignin biosynthesis did not sevefely affect the overall viability; instead, it promoted carbohydrate accumulation and enhanced the enzymatic hydrolysis of the remaining components of the cell wall, thus raising biofuel yield and decreasing processing costs.
Nevertheless, despite such successes in elucidating and manipulating lignin biosynthesis, the reverse genetic approach is not always straightforward. The complexity of monolignol biosynthetic pathways, the metabolic plasticity and the functional redundancy of the families of genes involved (e.g., there are more than 70 peroxidase genes in the Arabidopsis genome sequence) added uncertainties and complications to reverse genetic approaches aimed at exploring gene functions, elucidating lignification mechanisms, and biotechnological manipulation of lignin biosynthesis. Thus, additional approaches are desirable to dissect and manipulate plant lignification.
Regiospecific O-Methylation of Lignin Monomeric Precursors and Phenylpropenes.
S-adenosyl-L-methionine (SAM)-dependent methyltransferases are involved in the biosynthesis of a variety of small molecule compounds in plants, such as phenylpropenes, lignin monomeric precursors, flavonoids, isoflavonoids, alkaloids, and polyalcohols. Methylation essentially determines the specific physiological functions of the resultant molecules. In monolignol biosynthesis, the O-methylation of lignin monomeric precursors is catalyzed by two distinct types of O-methyltransferases, namely, caffeate/5-hydroxyferulate 3/5-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCoAOMT). COMT, a homodimer with a large subunit of 38-40 KDa, belongs to the plant type I methyltransferase family and does not require metal ions for catalysis. The enzymes from many plant species have been extensively characterized. It was originally recognized as being responsible for methylating caffeic acid and 5-hydroxyferulic acid, and lately was re-evaluated as predominating p-cinnamaldehyde and cinnamyl alcohol methylation. The enzyme displays very broad substrate-specificity in vitro, methylating a range of phenolics with propanoid tails bearing different functionalities (i.e., carboxylate, aldehyde and alcohol); but, for all substrates, it exhibited exclusive regiospecificity for meta (3 or 5)-hydroxylmethylation. The crystal structure of alfalfa COMT has been determined. Ternary complexes with the methyl donor SAM/SAH and substrate caffeic acid/5-hydroxyconiferaldehyde clearly revealed the structural basis for its substrate promiscuity and 3/5-OH specific methylation.
As consequence of the activities of lignin O-methyltransferases, monolignols and their monomeric precursors are methylated only at the meta-positions (i.e., 3-OH or 5-OH) of the phenyl rings (FIG. 1A). The para-hydroxyl position of lignin precursors is never methylated, pointing to the importance of the free para-hydroxyl of monolignol in lignin biosynthesis and polymerization. In fact, in all current lignin biosynthetic scenarios, the free para-hydroxyl of monolignol is implicated to be critical for monolignol dehydrogenation (FIG. 1B), for cross-coupling to form inter-unit linkages, and for glycosylation of monolignols for their storage, and perhaps transport (FIG. 1A). Consistently, our data showed that the phenolic compound bearing the methoxyl moiety at its para-position is inactive in coupling reaction to Gibbs' reagent and in forming in vitro synthetic lignin. Therefore, methylation of the para-hydroxyls (i.e., 4-OH) of monolignols should diminish their polymerization to form lignin.
Several O-methyltransferases characterized from a few plant species are able to catalyze the 4-O-methylation of a group of volatile compounds, the phenylpropenes isoeugenol, eugenol and chavicol. These allylphenols are structural analogs of monolignols, differing only in their propanoid tails. Particularly, isoeugenols closely resemble p-coniferyl alcohol. The characterized phenylpropene 4-OMTs include (iso)eugenol 4-O-methyltransferase (IEMT) from Clarkia breweri, eugenol and chavicol 4-O-methyltransferases (EOMT and CVOMT) from sweet basil, and two additional enzymes (SbOMT1 and SbOMT3) from sorghum (Baerson et al, unpublished data). Among them, IEMT from C. breweri shares more than 83% sequence identity at the amino acid level with caffeic acid 3-O-methyltransferase (COMT) from the same species, but exhibits distinct substrate preferences and regio-specificity for 4-hydroxyl methylation of phenylpropenes.
Based on sequence analysis, Pichersky and his colleague (Wang and Pichersky, Arch. Biochem. Biophys. 368:172-180 (1999) and Wang and Pichersky, Arch Biochem. Biophys. 349:153-160 (1998)) previously conducted rational mutagenesis on IEMT and COMT and demonstrated that reciprocally replacing strategic amino acid residues of IEMT and COMT could inversely switch both the substrate preference and regiospecificity of two enzymes to each other; i.e., substitution mutations converted IEMT from the 4-O-methylation of isoeugenol to the 3/5-O-methylation of caffeic acid, and switched COMT from the 3/5-O-methylation of caffeic acid to the 4-O-methylation of isoeugenol. These pioneering studies demonstrate the plasticity of these two closely related enzymes. However, their studies did not report any mutant enzymes with 4-O-methylation activity toward lignin monomeric precursors.
Directed Protein Evolution.
Numerous biochemical analyses suggested that the plasticity of proteins, yielding novel or altered functions, rests upon a few amino-acid substitutions. Recently, directed protein evolution has been broadly applied to engineer enzymes with novel functions or improved properties. Among the many sophisticated mutagenesis methods being developed, Gene Site Saturation Mutagenesis (GSSM) represents a very non-stochastic random mutagenesis approach. This comprehensive technique introduces minimally all possible single amino-acid substitutions (up to 19) into the targeted site via degenerate primers. Subsequently combining the single beneficial substitutions into one variant by combinatorial gene reassembly heightens the efficiency of this strategy. In addition to the site mutagenesis, another efficient way to evolve protein's function and property is through DNA family shuffling to create gene chimeras. Since the related enzymes are from the same family and share common folds, the chimeric polypeptides are likely to be functional because they can fold appropriately. One highly efficient DNA family chimeragenic method being developed is “Random Chimeragenesis on Transient Templates” (RACHITT) wherein one single-strand parental DNA is used as a transient template to guide the hybridization of the gene fragments from the homologous gene in the same family to create “mosaic” chimeras. Compared to other conventional in vitro recombination methods like “sexual PCR” gene shuffling and the “staggered extension process”, RACHITT generates high resolution recombinatory crossovers at high frequency in gene-family-shuffled libraries (averaging 14 crossovers per gene vs four or fewer using other DNA shuffling methods). Thus, it greatly expands the diversity of chimeric variants. The method has been used both for improving the enzyme catalytic efficiency and substrate specificity.